Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.
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References
1.
BaralleFE, GiudiceJ. Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol, 2017; 18:437–451.
2.
StanleyRF, Abdel-WahabO. Dysregulation and therapeutic targeting of RNA splicing in cancer. Nat Cancer, 2022; 3:536–546.
BaileyP, ChangDK, NonesK, et al.Genomic analyses identify molecular subtypes of pancreatic cancer. Nature, 2016; 531:47–52.
7.
JbaraA, LinK-T, StosselC, et al.RBFOX2 modulates a metastatic signature of alternative splicing in pancreatic cancer. Nature, 2023; 617:147–153.
8.
HayesGM, CarriganPE, BeckAM, et al.Targeting the RNA splicing machinery as a novel treatment strategy for pancreatic carcinoma. Cancer Res, 2006; 66:3819–3827.
9.
LuSX, De NeefE, ThomasJD, et al.Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. Cell, 2021; 184:4032–4047.e4031.
10.
KimJ, KooB-K, KnoblichJA. Human organoids: Model systems for human biology and medicine. Nat Rev Mol Cell Biol, 2020; 21:571–584.
11.
ToshimitsuK, TakanoA, FujiiM, et al.Organoid screening reveals epigenetic vulnerabilities in human colorectal cancer. Nat Chem Biol, 2022; 18:605–614.
12.
DriehuisE, van HoeckA, MooreK, et al.Pancreatic cancer organoids recapitulate disease and allow personalized drug screening. Proc Natl Acad Sci USA, 2019; 116:26580–26590.
13.
TuvesonD, CleversH. Cancer modeling meets human organoid technology. Science, 2019; 364:952–955.
14.
DhuriK, BechtoldC, QuijanoE, et al.Antisense oligonucleotides: An emerging area in drug discovery and development. J Clin Med, 2020; 9:2004.
15.
LiangX-h, ShenW, SunH, et al.Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat Biotechnol, 2016; 34:875–880.
16.
DowlingJJ. Eteplirsen therapy for Duchenne muscular dystrophy: Skipping to the front of the line. Nat Rev Neurol, 2016; 12:675–676.
17.
FrankDE, SchnellFJ, AkanaC, et al.Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy. Neurology, 2020; 94:e2270–e2282.
18.
HuaY, SahashiK, RigoF, et al.Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature, 2011; 478:123–126.
19.
BojSF, HwangCI, BakerLA, et al.Organoid models of human and mouse ductal pancreatic cancer. Cell, 2015; 160:324–338.
20.
Martinez-OrdoñezA, DuranA, Ruiz-MartinezM, et al.Hyaluronan driven by epithelial aPKC deficiency remodels the microenvironment and creates a vulnerability in mesenchymal colorectal cancer. Cancer Cell, 2023; 41:252–271.e259.
21.
LangnerHK, JastrzebskaK, CaruthersMH. Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras. J Am Chem Soc, 2020; 142:16240–16253. Caruthers, MH and Paul, S. US Patent 11,230,565 B2.
22.
MarascoLE, DujardinG, Sousa-LuísR, et al.Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy. Cell, 2022; 185:2057–2070.e2015.
23.
MaWK, VossDM, ScharnerJ, et al.ASO-based PKM splice-switching therapy inhibits hepatocellular carcinoma growth. Cancer Res, 2022; 82:900–915.
24.
WangZ, JeonHY, RigoF, et al.Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides. Open Biol, 2012; 2:120133.
25.
HuaY, VickersTA, OkunolaHL, et al.Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice. Am J Hum Genet, 2008; 82:834–848.
26.
MattisVB, EbertAD, FossoMY, et al.Delivery of a read-through inducing compound, TC007, lessens the severity of a spinal muscular atrophy animal model. Hum Mol Genet, 2009; 18:3906–3913.
27.
MattisVB, Tom ChangC-W, LorsonCL. Analysis of a read-through promoting compound in a severe mouse model of spinal muscular atrophy. Neurosci Lett, 2012; 525:72–75.
28.
TakenakaM, NoguchiT, InoueH, et al.The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem, 1986; 264:2363–2367.
29.
ChristofkHR, Vander HeidenMG, HarrisMH, et al.The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature, 2008; 452:230–233.
30.
ChristofkHR, Vander HeidenMG, WuN, et al.Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature, 2008; 452:181–186.
31.
WangZ, ChatterjeeD, JeonHY, et al.Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons. J Mol Cell Biol, 2011; 4:79–87.
32.
CartegniL, HastingsML, CalarcoJA, et al.Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2. Am J Hum Genet, 2006; 78:63–77.
33.
ClowerCV, ChatterjeeD, WangZ, et al.The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism. Proc Natl Acad Sci USA, 2010; 107:1894–1899.
34.
ScharnerJ, MaWK, ZhangQ, et al.Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides. Nucleic Acids Res, 2019; 48:802–816.
35.
RobertsTC, LangerR, WoodMJA. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov, 2020; 19:673–694.
36.
TiriacH, BelleauP, EngleDD, et al.Organoid profiling identifies common responders to chemotherapy in pancreatic cancer. Cancer Discov, 2018; 8:1112–1129.
37.
PriorTW. (2017). Chapter 4—Strategy for the molecular testing of spinal muscular atrophy. In: SumnerCJ, PaushkinS and KoCP (eds). Spinal Muscular Atrophy. Academic Press. pp 63–71.
38.
RaghavanS, WinterPS, NaviaAW, et al.Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer. Cell, 2021; 184:6119–6137 e6126.
39.
MiyabayashiK, BakerLA, DeschênesA, et al.Intraductal transplantation models of human pancreatic ductal adenocarcinoma reveal progressive transition of molecular subtypes. Cancer Discov, 2020; 10:1566–1589.
40.
BakerLA, TiriacH,, TuvesonDA. (2019). Generation and culture of human pancreatic ductal adenocarcinoma organoids from resected tumor specimens. In: SuGH (ed). Pancreatic Cancer: Methods and Protocols. Springer. New York: New York, NY. pp 97–115.
41.
IkedaY, TanakaT, NoguchiT. Conversion of non-allosteric pyruvate kinase isozyme into an allosteric enzyme by a single amino acid substitution. J Biol Chem, 1997; 272:20495–20501.
42.
IsraelsenWJ, DaytonTL, DavidsonSM, et al.PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells. Cell, 2013; 155:397–409.
43.
HuangL, HoltzingerA, JaganI, et al.Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell– and patient-derived tumor organoids. Nat Med, 2015; 21:1364–1371.
44.
El MarabtiE, Abdel-WahabO. Therapeutic modulation of RNA splicing in malignant and non-malignant Disease. Trends Mol Med, 2021; 27:643–659.
45.
NomakuchiTT, RigoF, AznarezI, et al.Antisense oligonucleotide–directed inhibition of nonsense-mediated mRNA decay. Nat Biotechnol, 2016; 34:164–166.
46.
DenichenkoP, MogilevskyM, CléryA, et al.Specific inhibition of splicing factor activity by decoy RNA oligonucleotides. Nat Commun, 2019; 10:1590.
47.
KriegAM. CpG still rocks! Update on an accidental drug. Nucleic Acid Ther, 2012; 22:77–89.
48.
KelleyKW, PașcaSP. Human brain organogenesis: Toward a cellular understanding of development and disease. Cell, 2022; 185:42–61.
49.
KelavaI, ChiaradiaI, PellegriniL, et al.Androgens increase excitatory neurogenic potential in human brain organoids. Nature, 2022; 602:112–116.
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